Characterization of Four Multidrug Resistance Plasmids Captured from the Sediments of an Urban Coastal Wetland.

Self-transmissible and mobilizable plasmids contribute to the emergence and spread of multidrug-resistant bacteria by enabling the horizontal transfer of acquired antibiotic resistance. The objective of this study was to capture and characterize self-transmissible and mobilizable resistance plasmids from a coastal wetland impacted by urban stormwater runoff and human wastewater during the rainy season. Four plasmids were captured, two self-transmissible and two mobilizable, using both mating and enrichment approaches. Plasmid genomes, sequenced with either Illumina or PacBio platforms, revealed representatives of incompatibility groups IncP-6, IncR, IncN3, and IncF. The plasmids ranged in size from 36 to 144 kb and encoded known resistance genes for most of the major classes of antibiotics used to treat Gram-negative infections (tetracyclines, sulfonamides, ß-lactams, fluoroquinolones, aminoglycosides, and amphenicols). The mobilizable IncP-6 plasmid pLNU-11 was discovered in a strain of Citrobacter freundii enriched from the wetland sediments with tetracycline and nalidixic acid, and encodes a novel AmpC-like ß-lactamase (blaWDC-1), which shares less than 62% amino acid sequence identity with the PDC class of ß-lactamases found in Pseudomonas aeruginosa. Although the IncR plasmid pTRE-1611 was captured by mating wetland bacteria with P. putida KT2440 as recipient, it was found to be mobilizable rather than self-transmissible. Two self-transmissible multidrug-resistance plasmids were also captured: the small (48 kb) IncN3 plasmid pTRE-131 was captured by mating wetland bacteria with Escherichia coli HY842 where it is seemed to be maintained at nearly 240 copies per cell, while the large (144 kb) IncF plasmid pTRE-2011, which was isolated from a cefotaxime-resistant environmental strain of E. coli ST744, exists at just a single copy per cell. Furthermore, pTRE-2011 bears the globally epidemic blaCTX-M-55 extended-spectrum ß-lactamase downstream of ISEcp1. Our results indicate that urban coastal wetlands are reservoirs of diverse self-transmissible and mobilizable plasmids of relevance to human health.

High Diversity of CTX-M Extended-Spectrum ß-Lactamases in Municipal Wastewater and Urban Wetlands.

The CTX-M-type extended-spectrum ß-lactamases (ESBLs) present a serious public health threat as they have become nearly ubiquitous among clinical gram-negative pathogens, particularly the enterobacteria. To aid in the understanding and eventual control of the spread of such resistance genes, we sought to determine the diversity of CTX-M ESBLs not among clinical isolates, but in the environment, where weaker and more diverse selective pressures may allow greater enzyme diversification. This was done by examining the CTX-M diversity in municipal wastewater and urban coastal wetlands in southern California, United States, by Sanger sequencing of polymerase chain reaction amplicons. Of the five known CTX-M phylogroups (1, 2, 8, 9, and 25), only genes from groups 1 and 2 were detected in both wastewater treatment plants (WWTPs), and group 1 genes were also detected in one of the two wetlands after a winter rain. The highest relative abundance of blaCTX-M group 1 genes was in the sludge of one WWTP (2.1 × 10(-4) blaCTX-M copies/16S rRNA gene copy). Gene libraries revealed surprisingly high nucleotide sequence diversity, with 157 new variants not found in GenBank, representing 99 novel amino acid sequences. Our results indicate that the resistomes of WWTPs and urban wetlands contain diverse blaCTX-M ESBLs, which may constitute a mobile reservoir of clinically relevant resistance genes.

High expression of CAI2, a 9p21-embedded long noncoding RNA, contributes to advanced-stage neuroblastoma.

Neuroblastoma is a pediatric cancer with significant genomic and biologic heterogeneity. p16 and ARF, two important tumor-suppressor genes on chromosome 9p21, are inactivated commonly in most cancers, but paradoxically overexpressed in neuroblastoma. Here, we report that exon γ in p16 is also part of an undescribed long noncoding RNA (lncRNA) that we have termed CAI2 (CDKN2A/ARF Intron 2 lncRNA). CAI2 is a single-exon gene with a poly A signal located in but independent of the p16/ARF exon 3. CAI2 is expressed at very low levels in normal tissue, but is highly expressed in most tumor cell lines with an intact 9p21 locus. Concordant expression of CAI2 with p16 and ARF in normal tissue along with the ability of CAI2 to induce p16 expression suggested that CAI2 may regulate p16 and/or ARF. In neuroblastoma cells transformed by serial passage in vitro, leading to more rapid proliferation, CAI2, p16, and ARF expression all increased dramatically. A similar relationship was also observed in primary neuroblastomas where CAI2 expression was significantly higher in advanced-stage neuroblastoma, independently of MYCN amplification. Consistent with its association with high-risk disease, CAI2 expression was also significantly associated with poor clinical outcomes, although this effect was reduced when adjusted for MYCN amplification. Taken together, our findings suggested that CAI2 contributes to the paradoxical overexpression of p16 in neuroblastoma, where CAI2 may offer a useful biomarker of high-risk disease.